Substitution of the native variable region-1 (VR1/37-loop) of thrombin by the corresponding VR1 of tissue-type plasminogen activator (thrombin-VR1(tPA)) increases the rate of inhibition by plasminogen activator inhibitor type 1 (PAI-1) by three order ...

Substitution of the native variable region-1 (VR1/37-loop) of thrombin by the corresponding VR1 of tissue-type plasminogen activator (thrombin-VR1(tPA)) increases the rate of inhibition by plasminogen activator inhibitor type 1 (PAI-1) by three orders of magnitude, and is thus sufficient to confer PAI-1 specificity to a heterologous serine protease. A structural and kinetical approach to establish the function of the VR1 loop of t-PA in the context of the thrombin-VR1(tPA) variant is described. The crystal structure of thrombin-VR1(tPA) was resolved and showed a conserved overall alpha-thrombin structure, but a partially disordered VR1 loop as also reported for t-PA. The contribution of a prominent charge substitution close to the active site was studied using charge neutralization variants thrombin-E39Q(c39) and thrombin-VR1(tPA)-R304Q(c39), resulting in only fourfold changes in the PAI-1 inhibition rate. Surface plasmon resonance revealed that the affinity of initial reversible complex formation between PAI-1 and catalytically inactive Ser195-->Ala variants of thrombin and thrombin-VR1(tPA) is only increased fivefold, i.e. KD is 652 and 128 nM for thrombin-S195A and thrombin-S195A-VR1(tPA), respectively. We established that the partition ratio of the suicide substrate reaction between the proteases and PAI-1 was largely unaffected in any variant studied. Hirugen allosterically decreases the rate of thrombin inhibition by PAI-1 2.5-fold and of thrombin-VR1(tPA) 20-fold, by interfering with a unimolecular step in the reaction, not by decreasing initial complex formation or by altering the stoichiometry. Finally, kinetic modeling demonstrated that acylation is the rate-limiting step in thrombin inhibition by PAI-1 (k approximately 10(-3) s(-1)) and this kinetic block is alleviated by the introduction of the tPA-VR1 into thrombin (k>1 s(-1)). We propose that the length, flexibility and different charge architecture of the VR1 loop of t-PA invoke an induced fit of the reactive center loop of PAI-1, thereby enhancing the rate of acylation in the Michaelis complex between thrombin-VR1(t-PA) and PAI-1 by more than two orders of magnitude.